Mobile Phase pH Research Articles

Simulation of Adsorption Process of l-Tryptophan on Mixed-Mode Resin HD-1 with Combined Physical Adsorption and Ion Exchange.

The mass-transfer process of l-tryptophan (l-Trp) in the hydrophobic interaction/ion-exchange mixed-mode resin HD-1 particles and fixed bed was studied experimentally and theoretically. The adsorption kinetics of l-Trp in single-component and multicomponent adsorption systems was investigated under different pH conditions. The co-adsorption of sodium ions (Na+) and l-Trp anions was found to be negligible. A modified liquid-film linear driving force model considering the physical adsorption of l-Trp zwitterions and anions as well as ion exchange of l-Trp cations was proposed. The dissociation equilibria of l-Trp molecules and functional groups on the resin were introduced in the model. The model could well fit the kinetic adsorption curves of l-Trp at different pH values. The presence of Na+ and the impurity amino acid l-glutamic acid (l-Glu) did not significantly affect the mass-transfer rate of l-Trp. The dynamic adsorption processes of l-Trp under different pH and concentration conditions were studied. A modified transport-dispersive model considering axial diffusion, liquid-film mass transfer, and a combined physical adsorption and ion-exchange equilibrium was established, which could predict the adsorption breakthrough curves of l-Trp well. During the dynamic adsorption process, the pH of mobile phase in the fixed bed changed with changing the l-Trp concentration in the mobile phase. l-Trp was well separated from Na+ and l-Glu with the purity of l-Trp higher than 99%, the recovery rate higher than 95%, and a concentration of 4.69 × 10–3 mol/L. The elution chromatographic peaks of l-Trp, l-Glu, and Na+ and the pH of the outlet solution were predicted satisfactorily.

Implementation of QbD Approach to the Development of Chromatographic Methods for the Determination of Complete Impurity Profile of Substance on the Preclinical and Clinical Step of Drug Discovery Studies.

The purpose of this work was to demonstrate the use of the AQbD with the DOE approach to the methodical step-by-step development of a UHPLC method for the quantitative determination of the impurity profile of new CPL409116 substance (JAK/ROCK inhibitor) on the preclinical and clinical step of drug discovery studies. The critical method parameters (CMPs) have been tested extensively: the kind of stationary phase (8 different columns), pH of the aqueous mobile phase (2.6, 3.2, 4.0, 6.8), and start (20–25%) and stop (85–90%) percentage of organic mobile phase (ACN). The critical method attributes (CMAs) are the resolution between the peaks (≥2.0) and peak symmetry of analytes (≥0.8 and ≤1.8). In the screening step, the effects of different levels of CMPs on the CMAs were evaluated based on a full fractional design 22. The robustness tests were established from the knowledge space of the screening step and performed by application fractional factorial design 2(4−1). Method operable design region (MODR) was generated. The probability of meeting the specifications for the CMAs was calculated by Monte-Carlo simulations. In relation to literature such a complete AQbD approach including screening, optimization, and validation steps for the development of a new method for the quantitative determination of the full profile of nine impurities of an innovative pharmaceutical substance with the structure-based pre-development pointed out the novelty of our work. The final working conditions were as follows: column Zorbax Eclipse Plus C18, aqueous mobile phase 10 mM ± 1 mM aqueous solution of HCOOH, pH 2.6, 20% ± 1% of ACN at the start and 85% ± 1% of ACN at the end of the gradient, and column temperature 30 °C ± 2 °C. The method was validated in compliance with ICH guideline Q2(R1). The optimized method is specified, linear, precise, and robust. LOQ is on the reporting threshold level of 0.05% and LOD at 0.02% for all impurities.

Chemistry in a Bottle: Ester Formation in Acidified Mobile-Phase Solvents

Acidified solvents are commonly used in reversed-phase liquid chromatography (RPLC) to adjust mobile-phase pH and the ionization state of ionogenic compounds. When methanol-containing solvents are acidified with formic or trifluoroacetic acids, esters can be formed at chromatographically relevant rates, affecting baseline quality, retention, and selectivity. Understanding these effects is the first step toward avoiding the problems that can be caused by esterification in the bottle.

Retention behaviour of analytes in reversed-phase high-performance liquid chromatography-A review.

The understanding of principles that drive the separation in reversed-phase chromatography plays an important role in the prediction of the elution of solutes in RP-HPLC. The separation in RP-HPLC is based on the principle of adsorption and partition. In addition, the logP value, the pKa value of the drug and chromatographic parameters like mobile phase pH, buffer concentration, organic modifier and mobile phase additives also influence the retention and selectivity of the analyte. It was found that hydrophobic, electrostatic, hydrogen bonding and other specific interactions between the stationary phase and the solutes, along with the hydrophobicity of an analyte molecule (logP), modify the retention behaviour of the analytes. This article gives special attention to the influence of ionization and ion interaction on the separation of analytes. The drug molecules with different logP values containing protonated and deprotonated acids, bases and zwitterions are selected as examples and this article addresses various issues related to the method development, relationships between analyte retention and mobile phase pH and the pKa value of the analyte. The advances in this regard, with highlights on topics such as mechanisms of retention and various factors that influence the retention behaviour of analytes, are also updated with suitable examples.

Micellar and sub-micellar chromatography with a cocamidopropyl betaine surfactant

We have coated a typical C18 column with the pH cationic charge controllable zwitterionic synthetic surfactant, cocamidopropyl betaine (CAPB), in order to generate a mixed mode reversed phase weak ion exchange column. As determined by the Thomas model, the column has an adsorbed surfactant capacity of 0.557 mmoles. The addition of 8.8 × 10–4 M CAPB to the totally aqueous mobile phase ensured stability of the surfactant on the column and permitted separation of the four component sulfonamide mixture with micellar liquid chromatography (MLC) in under 11 min. Comparatively, with a dilute H2SO4 mobile phase with no CAPB, the separation time for the sulfonamide mixture reached an excessive run time of an hour on the bare C18 chains. With CAPB in the mobile phase (no organic solvent present), a seven component sulfonamide mixture could be separated in less than 45 min. A five component short chain carboxylic acid mixture, separated in 20 min, was used to examine the ion exchange character of the column in pH environments of 2.3 and 4.6. Three phase MLC equilibrium analysis was also done in these pH environments with the sulfa drug and carboxylic acid mixtures to determine partition coefficients. Finally, a quite high molecular weight (70,000) anionic polystyrene sulfonate polymer was characterized by MLC with only CAPB and variable pH mobile phases; the optimal pH was determined to be 5.6. A totally aqueous mobile phase without CAPB was not suitable for profiling this polymer.

VALIDATION OF STABILITY INDICATING RP-HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF JANUMET XR (SITAGLIPTIN/METFORMIN HCL EXTENDED-RELEASE FIXED DOSE COMBINATION (FDC) CONTENT IN BULK AND PHARMACEUTICAL DOSAGE FORM

A simple, accurate, precise and rapid stability indicating reverse phase High performance chromatography method was used for estimation of Sitagliptin/metformin HCl ER tablets in bulk and fixed-dose combination solid oral dosage form. The proposed analytical method has been validated for specificity, Linearity, Accuracy, Precision and Robustness. The chromatography was achieved in a Avantor, ACE C18 (Length 150 x Diameter 4.6mm Particle size 5µm) column with gradient flow. The optimal chromatographic condition consisted of mobile phase pH 3.5 at a flow rate of 1.2 mL/min, with a column temperature of 35°C, run time 17 minutes and detector wavelength of 210 nm (Sitagliptin& Propyl gallate and Metformin HCl).

Effect of mobile phase pH on liquid chromatography retention of mepartricin related compounds and impurities as support to the structural investigation by liquid chromatography–mass spectrometry

Mepartricin is a semisynthetic polyene macrolide with antifungal and anti-protozoal activities, and it is widely used for the treatment of benign prostatic hyperplasia. Mepartricin is produced by synthetic methyl esterification of the more toxic partricin, and its activity is due to a complex of related compounds. Among them, the main ones are mepartricin B and mepartricin A which are characterized by the presence of a primary and a secondary amine group, respectively. In this work a previously reported HPLC-UV method was properly modified to make it MS-compatible. The selected conditions entail the use of a C18 reverse phase column, and a mobile phase composed by ammonium formate and acetonitrile, with the addition of heptafluorobutyric acid as modifier. The developed method was applied to the characterization of a mepartricin reference standard and a mepartricin experimental batch. All the UV responding peaks, 30 for the standard and 21 for the experimental batch, were successfully detected by MS, allowing to define their m/z values and acquire their fragmentation spectra. For the structural elucidation of isobaric species and, in particular, the identification of toxic partricin-related impurities, the presence of differently ionisable chemical groups was considered, as partricins contain free caboxy-groups, while mepartricins represent their estherified counterparts. A deep study of the effect of mobile phase pH on the chromatographic retention of partricin and mepartricin related compounds was performed in the pH range 2.5–6.5. This study allowed to successfully cluster all the detected species and asses, in the considered batch, the absence of other partricin-related impurities in addition to partricin B and partricin A.

Determination of Atogepant Using RP-HPLC in Bulk and Table ts

Context: Around one in seven individuals worldwide suffers from migraine, a highly prevalent and chronic neurological disease. In the prevention and treatment of migraines, 'gepants' are a class of receptor antagonist molecules that block calcitonin gene-related peptide (CGRP) receptors. Atogepant (ATO) is the first and only oral CGRP receptor antagonist (gepant) that was developed exclusively for the prevention and treatment of episodic migraines. Aim A stability-indicating reverse phase high performance liquid chromatography (RP-HPLC) method for the determination of ATO has been developed and validated in bulk and tablet dosage forms. Materials and Methods The chromatographic analysis was carried out on a Waters C18 Column with 250 mm × 4.6 mm and a particle size of 5 μm, using an isocratic mobile phase of Phosphate Buffer pH 3.6: Methanol: 0.5% Formic acid 60:38:2 v/v at a flow rate of 0.8 mL/min, and the eluents were monitored at an isosbestic point of 217 nm. Results The specificity, precision, accuracy, linearity, and robustness of the proposed method were all validated according to the ICH standards. Forced degradation studies confirmed the method's stability-indicating nature. ATO had retention time of 3.58 min. The current method was found to be accurate, precise, and sensitive. ATO linearity was achieved between 105 and 315 μg/mL, while LOD and LOQ were found to be 4.33 and 14.44 μg/mL, respectively. Conclusion As a result, the suggested RP-HPLC method for the quantification of ATO was reliable, reproducible, accurate, and sensitive.

A Validated RP-HPLC Method for Simultaneous Determination of Cefixime and Clavulanic Acid Powder in Pediatric Oral Suspension.

A new reverse phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the simultaneous estimation of pediatric oral powder formulation containing cefixime (CFX) and clavulanic acid (CVA). In this research, an analytical C18 (4.6 mm × 25 cm), 5 μm column was used for chromatographic separation with a mixture of methanol and water containing disodium hydrogen phosphate in ratio of 20 : 80 v/v as the mobile phase (pH 5.5 adjusted with orthophosphoric acid) at a flow rate of 1.0 mL/min. The detecting wavelength and run time were 220 nm and 15 min, respectively. Moreover, the column temperature was maintained at 30°C. The analytical method was validated prior to meeting the conditions specified by International Conference on Harmonization (ICH) and the parameters were specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision, robustness, and solution stability. The calibration curve was found to be linear between the concentration ranges of 0.024–0.036 mg/mL and 0.032–0.048 mg/mL for CFX and CVA, respectively. Furthermore, the LOD and LOQ of CFX were 0.0008 and 0.0025 μg/mL, respectively. Accordingly, LOD and LOQ of CVA were 0.0021 and 0.0065 μg/mL, respectively. The accuracy of the optimized method was examined by recovery studies and the mean recovery was observed to be 98.96% and 99.05% for CFX and CVA, respectively, at 100% spiked level. The repeatability testing for both standard and sample solutions revealed that the method is precise within the acceptable range and the %RSD of the precision was <2%. In addition, the findings of specificity, linearity, accuracy, precision, robustness, LOD, LOQ, and solution stability studies of both CFX and CVA were within the criteria of acceptable limit as well.

Quantitative evaluation of reversed-phase packing material based on calcium carbonate microspheres modified with an alternating copolymer

Considering the vulnerability of silica gel to alkaline mobile phases, a highly alkaline stable stationary phase for HPLC is required to separate basic compounds with high separation efficiency. To address this issue, we have developed a high alkaline stable packing material (CaCO3-PMAcO) based on mesoporous calcium carbonate microspheres modified with poly(maleic acid-alt-1-octadecene). In this study, we report further investigation of the separation performance of CaCO3-PMAcO column by systematically evaluating the effects of particle size and chromatographic conditions. Based on the theory of the van Deemter equation, the separation efficiency was related to the size of CaCO3-PMAcO particles (2.9 - 5.7 µm). The evaluation of thermodynamics of retention by changing the column temperature from 20 °C to 45 °C implied that the retention mode was dominated by hydrophobic interaction associated with the exothermic enthalpy changes (-11.1 to -12.5 kJ/mol). The results of column selectivity tests revealed that the CaCO3-PMAcO column had hydrophobic selectivity comparable to C18 silica gel columns (αP/B; CaCO3-PMAcO column: 1.53, C18 column: 1.69), and higher shape/steric selectivity (αTri/Ter; CaCO3-PMAcO column: 1.56, C18 column: 0.955). In practice, the CaCO3-PMAcO column could be applied to the separation of not only alkylbenzenes and polycyclic aromatic hydrocarbons, but also to basic tricyclic antidepressants by using an alkaline mobile phase (pH 12).

A New Strategy for the Software-Assisted LC Separations of Ketoconazole and Its Impurities.

Analytical quality by design and the use of dissimilar chromatographic systems can be employed to accelerate chromatographic separations. Herein, a software (S-Matrix)-assisted platform was used to proficiently screen, optimize and select the optimal parameters for the chromatographic separation of ketoconazole and its related impurities. This approach evaluated the various chromatographic parameters in a stepwise manner based on the statistical tools and provided an in-depth understanding of the critical parameters influencing the peak selectivities and separations. It was demonstrated that dissimilar conditions, such as different stationary phases, mobile phase pH and organic modifiers (i.e., critical method variables), can improve the peak resolution, while the critical quality attributes can provide conditions appropriate for quantification purposes via a quality target analytical method. Furthermore, an orthogonal method was established to support the primary method. The orthogonality between the two methods was defined by the correlation matrix between the two systems using the Pearson correlation coefficient and was found to be 0.12. Using the optimized method, the primary method was validated as per International Council for Harmonization in the range of 0.05-1.0% for impurities and 80.0-120.0% for ketoconazole, thereby indicating the suitability of the method for use in quality control laboratories.

Evaluation of the Base Stability of Hydrophilic Interaction Chromatography Columns Packed with Silica or Ethylene-Bridged Hybrid Particles

Stability as a function of mobile phase pH is an important consideration when selecting a chromatographic column. While the pH stability of reversed-phase columns is widely studied, there are relatively few reports of the stability of hydrophilic interaction chromatography (HILIC) columns. We evaluated the stability of silica and ethylene-bridged hybrid HILIC columns when used with mobile phases containing basic buffers. The predominant mode of column degradation observed in our studies was a decrease in efficiency due to voiding, resulting from the hydrolysis of the silica particles. Associated with this were increases in tailing factors. Retention factor changes were also noted but were smaller than the efficiency losses. The dependence of the rate of efficiency decrease on the key variables of temperature, mobile phase pH and water content were studied for an unbonded silica column. The effect of the acetonitrile concentration on the pH of the mixed aqueous/acetonitrile mobile phases was also investigated. Using conditions found to cause a 50% decrease in efficiency after approximately five hours of exposure to the basic solution, we evaluated eight different commercially available HILIC columns containing silica or ethylene-bridged hybrid particles. The results show large differences between the stability of the silica and ethylene-bridged hybrid particle stationary phases, with the latter exhibiting greater stability.

Combination of high performance liquid chromatography and flame atomic absorption spectrophotometry using a novel nebulizer interface supported T shaped slotted quartz tube for the determination of Vitamin B12

A novel nebulizer interface (NI) was proposed to combine high performance liquid chromatography (HPLC) and flame atomic absorption spectrophotometer (FAAS). A glass concentric nebulizer was linked to T-shaped slotted quartz tube (T-SQT) using a tubing to transfer the liquid solution eluted from the chromatographic system into the atomization region of FAAS system. T-SQT was also used to intensify the interaction of atoms with the hollow cathode lamp light. Vitamin B12 was selected as an analyte to show the applicability of the new hyphenated system. After optimizing some parameters such as mobile phase flow rate and pH, nebulizer gas flow rate, T-SQT height and injection volume, linear range for the analyte was determined between 4.7 and 92 mg/kg as Co. Limit of detection (LOD) and limit of quantitation (LOQ) for the HPLC-NI-T-SQT-FAAS system were calculated to be 1.6 and 5.3 mg/kg as Co, respectively. Recovery studies were also conducted to verify the accuracy and applicability of the developed method for vitamin tablets and excellent percent recovery results (~ 100%) with low standard deviation values were obtained when matrix-matching calibration strategy was performed for each vitamin tablet. A successful separation and detection of the analyte was achieved within 3.0 min that offers high sample throughput. Two different vitamin tablets were analyzed by the optimized hyphenated system. The developed method also provides low usage of sample solution in contrast to conventional nebulizer in the FAAS system.

Determination of diazepam in human plasma by developed and validated a high-performance liquid chromatographic ultraviolet method

Background and Aims: Diazepam is accepted as a safer drug to medicate in many serious cases, acting as an anticonvulsant, an anxiolytic and a treatment for many types of poisoning. Monitoring it is important in achieving successful treatment and reducing the risk of toxic effects. In this study, it is aimed to develop and validate a sensitive, repeatable, and reliable method based on high-performance liquid chromatographic analysis for the determination in human plasma. Methods: Separation was carried out using a reverse-phase C18 column (4.0 mm x 150 mm, 3 μm) at 30 °C. The solution was prepared with a 10 mM phosphate buffer and acetonitrile (1:1, v/v) was employed as a mobile phase at the isocratic flow with 0.5 mL/min rate. Quantification was applied at 230 nm. A solid-phase extraction method was established and optimized, which was then used in the preparation of the plasma (0.5 mL) samples to the analysis. Results: The method was found to be linear (r2 = 0.9805) between 100 and 1200 ng/mL. The analysis run was ≤12 min. Intraday and inter-day accuracy were found between -5.78 and 5.93 and precision was ≤1.82%. The limit of detection and quantification were calculated as 20.42 and 61.86 ng/mL, respectively. Recovery was found between the range of 95.12% and 106.83%. The method was determined to be robust according to changes in UV, mobile phase organic solvent content, mobile phase pH, column temperature, and operator. Conclusion: This simple, sensitive and reliable method is suggested for accredited-reference laboratories working on the therapeutic drug monitorization and/or overdose-toxicological quantified analysis of diazepam in human plasma.

A STUDY ON THE RETENTION BEHAVIOR OF SOME IMIDAZOLE ANTIMYCOTIC DRUGS IN ACETONITRILE-WATER BINARY MIXTURES

Objective: In this study, the thermodynamic protonation constant ((_s^s)〖pK〗_a ) of antifungal azole derivative miconazole and tioconazole were investigated by the reversed-phase liquid chromatography (RPLC) method. Although classical stationary phases are the most commonly used column-packed materials in the analyses of these compounds by the RPLC method, the determination of the pKa value of miconazole and tioconazole with a cyano-based column has not been reported so far. Material and Method: Chromatographic behavior of the selected compounds was determined with Ultra Cyano column (Restek®, 150×3.0 mm I.D, 3µm) which is compatible with hydrophobic compounds. The dependence of the retention times on the acetonitrile content of the mobile phase and the effect of the mobile phase pH on the chromatographic retention was determined for each compound by changing the pH in the range of 3.0-7.50. Result and Discussion: The (_s^s)〖pK〗_a values were determined by a nonlinear regression program. The thermodynamic aqueous pKa ((_w^w)〖pK〗_a ) value of the studied compounds was calculated from the (_s^s)〖pK〗_a value by using the macroscopic parameters (dielectric constant and mole fraction) that play an important role in the solvent properties. The calculated (_w^w)〖pK〗_a values were compared with literature values.

STABILITY INDICATING HPLC METHOD FOR ESTIMATION OF ELIGLUSTAT TARTRATE

For determination of eliglustat tartrate, a new, specific, simple, accurate (recovery) and precise stability indicating HPLC method has been developed and validated according to ICH Q2(R1) guidelines. The HPLC method was developed using acetonitrile: 0.1 % orthophosphoric acid 40:60 (V/V) as mobile phase, with pH of mobile phase adjusted to 3.0. The column used was HiQSil C-18 (250 mm x 4.6 mm, 5 μm) as stationary phase at a flow rate of 0.7 mL min-1. Retention time for eliglustat tartarate was found to be 5.9 min. The eluted compounds were detected using a PDA detector. The drug was subjected to stress testing as per ICH Q1A (R2) guideline. The degradation products did not interfere with the retention time of eliglustat. The calibration curve was found to be linear over a concentration range of 10-50 µg mL-1. For the calibration plots, R2 was found as 0.9995, indicating good linear relationship. The accuracy of the developed method was in the range of 99.87-100.62 %. The limit of detection of eliglustat was found to be 0.23 µg mL-1 and limit of quantification was 0.68 µg mL-1.

Rapid and sensitive simultaneous separation and electrochemical detection of tetracaine hydrochloride and oxymetazoline hydrochloride in pharmaceutical formulations via core-shell reversed-phase liquid chromatography

Tetracaine hydrochloride (TCH) is a nasal anesthetic and oxymetazoline hydrochloride (OZH) is a nasal decongestant. A moderate to acute overdosage of OZH and TCH can lead to mydriasis, nausea, cyanosis, tachycardia, dyspnoea, cardiovascular failure, disorientation, seizures, and even death. Liquid chromatography (LC) has been mainly utilized for the individual determination of either TCH or OZH; however, there is a need for rapid and efficient methods for simultaneous analysis in pharmaceutical formulations and aqueous samples. This study highlights the use of the fast and efficient separation capabilities of core-shell silica particles in liquid chromatography (LC) for the simultaneous determination of TCH and OZH using UV detection and the enhanced selectivity afforded by electrochemical detection at a boron-doped diamond (BDD) electrode. Rapid reversed-phase (RP) separation and detection of OZH and TCH in nasal spray and eye drops was achieved within 45 s using a poroshell 120 EC-C18 column, by adjusting the ratio of organic solvent, mobile phase pH, detection potential and mobile phase flow rate. Sensitivity was compared using ultraviolet (UV) detection at 280 nm, and ECD at + 1.3 V with detection limits of 40 and 70 nM for TCH and OZH, respectively. The developed rapid method was utilized successfully in the analysis of pharmaceutical formulations, where the estimated levels of TCH and OZH in these formulations are in agreement with the specified values outlined by the manufacturers.

Development of an Analytical Method for Determination of Related Substances and Degradation Products of Cabotegravir Using Analytical Quality by Design Principles.

Cabotegravir is one of the newly approved human immunodeficiency virus (HIV) integrase enzyme inhibitors used for the prevention and treatment of HIV infection. It is the first approved long-acting injectable antiretroviral therapy for HIV and is also very effective in combination with rilpivirine, a non-nucleoside reverse transcriptase inhibitor. Therefore, future drug development involving cabotegravir can be expected. We developed an ultrahigh performance liquid chromatography (UHPLC) method compatible with mass spectrometry for the determination of eight cabotegravir impurities. The described method is able to differentiate cabotegravir and its related substances as well as its degradation products. Analytical quality by design principles were used for method development. The method is robust within the defined method operable design region: flow rate = 0.32–0.40 mL/min; column temperature = 30–40 °C; pH of mobile phase A = 3.25–3.75, and the final percent of acetonitrile in gradient = 50.0–60.0%. Inside the method operable design region, a working optimal point was selected: pump flow rate = 0.36 mL/min; column temperature = 35 °C; pH of mobile phase A = 3.5, and final percent of acetonitrile in gradient = 55%. Method validation was performed, and the following parameters were verified: accuracy, repeatability, linearity, response factors, detection limit, and quantification limit. All method validation results were within selected criteria. The presented method could be used for the development of new pharmaceutical products based on cabotegravir.

Identification, Characterization, and the Determination of Process-related Impurities in Minodronic Acid Bulk Drug

Background: Minodronic acid (MA) is a third-generation nitrogen-containing heterocyclic bisphosphonate used to treat osteoporosis. In the process of starting materials research and preparation, the key intermediate impurities and degradation impurities have a great impact on the quality control of the drug. Objective: A sensitive, reliable, high-performance liquid chromatography (HPLC) method was developed and validated for the quantitative determination of MA and its related impurities (a total of 6 compounds, including 2 new impurities). Methods: The separation was achieved on an InertSustain ODS-4 C18 (250 mm × 4.6 mm, 5 μm) column using the mixture of 0.01 mol/L sodium pyrophosphate and 1 mmol tetrabutylammonium phosphate (the mobile phase pH was adjusted to 7.80 by phosphonic acid). Results: The quantitative analytical method was fully validated with respect to linearity (r &gt; 0.999), sensitivity (limit of detection &lt; 35 ng/mL), precision, accuracy (the recovery was between 98.7% and 104.2%), and robustness. Six process-related impurities in Minodronic Acid (MA) bulk drug were determined by high-performance liquid chromatography (HPLC). Furthermore, except for two starting materials, other four impurities were identified and characterized as 2-(imidazo[1,2-a] pyridin-3-yl) ethyl acetate (Imp-C), 2-(imidazo [1,2-a] pyridin- 3- yl)acetic acid (Imp-D), 3-(2-hydroxy-2,2- diphosphonoethyl)-4H-imidazo [1,2-a] pyridine -4- oxide (Imp-E) and 2,5- Dihydroxy- 3,6-bis(imidazo[1,2-a] pyridine-3-yl methyl) -2,5-dioxo- 1,4,2,5- dioxoDiphosphonium-3,6-diyl) bisphosphonic acid (Imp-F) using liquid chromatograph- mass spectrometer (LC-MS), MS/MS, Infrared Radiation and Nuclear Magnetic Resonance spectroscopy (1H-NMR and 13C-NMR). To the best of our knowledge, two of them (Imp- E and Imp-F) are new compounds and have not been reported previously. Conclusion: The HPLC method was developed and optimized, which could be applied for quantitative detection of the impurities, and further quality evaluation of MA.

Generic reversed‐phase ultra‐high‐pressure liquid chromatography methodology developed by using computer‐assisted modeling for streamlined performance evaluation of a wide range of stationary phase columns

AbstractSelection of column and mobile phase eluent/buffer is a crucial step in chromatographic method development. The introduction of new column technologies requires a continuous performance evaluation enabling the deployment of improved chromatographic screening workflows. Consequently, the development of a universal method that could be used across different column chemistries and mobile phase pH ranges would be highlighted as advantageous. Herein, we introduce a single generic probe developed via software‐based modeling that delivers excellent results for the separation of a standard multicomponent mixture across a wide range of stationary phases under different pH and temperature conditions. This approach has been applied to interrogate the performance and stability of multiple columns (C18, Cyano, Phenyl Hexyl, PFP, Polar‐embeded, and other stationary phases) using an automated ultra‐high‐pressure liquid chromatography screening system illustrating its practicality and effectiveness.